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J Neurosci Methods. 2012;206(2):200-4. doi: 10.1016/j.jneumeth.2012.02.023. Epub 2012 Mar 10.

Rapid expression profiling of brain microvascular endothelial cells by immuno-laser capture microdissection coupled to TaqMan(®) low density array.

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1
Blood-Brain Barrier Laboratory, Department of Cell Biology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030, United States.

Abstract

Immuno-laser capture microdissection (immuno-LCM) enables highly selective retrieval of designated cell populations from their in situ locations in complex tissue like the brain. However, the amount of tissue acquired by immuno-LCM is extremely limited, and the RNA purification, amplification and labeling steps necessary for expression analysis by hybridization microarray are tedious and time consuming. This report therefore describes a protocol in which these RNA steps are eliminated altogether, yet allows for global gene profiling. Specifically, immuno-LCM tissue was solubilized and the extract directly subjected to reverse transcription to generate cDNA. Pre-amplification of cDNA was performed next, and then relative expression of 96 different immune-related genes simultaneously determined by quantitative real-time PCR using a microfluidic card TaqMan(®) Low Density Array (TLDA). This protocol was highly reproducible and extremely sensitive, demonstrating high correlation of raw Ct values among both technical and biological replicate samples when using only 1/32 of total pre-amplified cDNA obtained from as little as 500 LCM 'shots.' As this abridged protocol takes only approximately 7h from LCM tissue acquisition to analysis by TLDA, it can prove a very effective tool for both screening and validation purposes when investigating gene regulation in health and disease of the nervous system and other tissues.

PMID:
22425714
PMCID:
PMC3327762
DOI:
10.1016/j.jneumeth.2012.02.023
[Indexed for MEDLINE]
Free PMC Article
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